The specific aim for this project is to analyze the structures and functions of the multiple protein isoforms of the recently described polyphosphatidyl inositol 5-phosphatase, SHIP. This protein is expressed in hematopoietic cells, and associates with characterized signaling molecules following stimulation of specific cell receptors. Although its function is not yet known, its expression in myeloid progenitor cells and phosphorylation and association with Shc following M-CSF treatment of these cells suggests a role in the regulation of myeloid hematopoiesis. Our strategy is divided into three sections. First, we are determining the cDNA structure of several SHIP isoforms detected in myeloid cells, using RT-PCR and low-stringency screening of cDNA libraries. We will analyze the functional role of SHIP proteins in vitro by transfecting isoforms of SHIP into cultured cells, and examining there cells for differences in morphology and growth characteristics as well as for alterations in the M-CSF signaling pathway. Finally, we will determine which SHIP isoforms are present at various times in myeloid hematopoiesis, using in vitro differentiation of embryonic stem cells. This will provide critical information toward our goal to produce a SHIP -/- mouse.